JP2957301B2 - Viable cell count / species identification system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the number of viable bacteria and for identifying the species thereof, and an apparatus used for carrying out the method. More specifically, the present invention uses a specific selective medium to grow only a specific bacterium selected from the group consisting of Escherichia coli, Salmonella, and Staphylococcus aureus in a specimen, and is generated from a culture of the bacterium. The present invention relates to a method and an apparatus for measuring the number of viable bacteria and identifying bacterial species by capturing fluorescence.

[0002]

2. Description of the Related Art Conventionally, identification of microorganism species has been carried out by using Berge (Ber
gey) by examining the morphological and physiological properties of the cultures. on the other hand,
The number of viable bacteria was 10 ^-1 , 10 ^-2 , ...,
After 10- ^n- fold dilution, a certain amount of the diluted solution was spread and inoculated on an agar plate medium, cultured for a certain period of time (for example, 24-48 hours), and then counted for the number of colonies that appeared on the agar plate. It was determined by multiplying by the dilution factor.

[0003] However, such a completely manual microbial testing method requires a testing period of 2 to 5 days, considerable skill, and may cause measurement differences between technicians or inspectors. Are known. In addition, the cost of testing is very high due to the use of large amounts of medium and dishes and the high labor costs of skilled technicians.

[0004] Microbial tests such as identification of microbial species and measurement of the number of viable bacteria are indispensable in departments such as clinical tests, food tests and pharmaceutical tests, and there is a high need for speeding up, labor saving and automation. In particular, the clinical testing department is developing various types of automation equipment. For example, a plater, which smears the sample solution from the center outward, is cultured on an agar plate medium rotating at 50 rpm, and the smeared agar medium is cultured, and the plate medium on which the colonies are formed is counted with a He-Ne laser. Spiral System Inc., a system containing various substrates, such as Auto Microbic System, which can automatically identify microbial species by colorimetry and perform drug susceptibility tests by turbidimetric absorption, MS-II, Avantage, etc. Dynatech's MIC-2000 system using microplates instead of systems and cartridges has been developed mainly in the United States.

However, most of these instruments are used only for microbial tests such as urinary tract infection, and cannot be measured when samples are often turbid, such as in food tests. Yes, and sufficient accuracy could not be obtained. In addition, any method requires a culture time of 24 to 48 hours, so that rapid measurement is difficult. For this reason, it has not reached the stage where it can sufficiently respond to the urgent needs of the site. Research on these automatic devices and measuring methods has been actively conducted in the fields of clinical medicine and medical engineering, but very few have been put to practical use.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for measuring the number of viable bacteria which can be used in a wide range of fields, is highly accurate, is low in cost, and requires only a few hours for inspection.
An object of the present invention is to provide a method for identifying a bacterial species and an apparatus used for performing the method.

[0007]

In order to achieve the above object, according to the present invention, a microorganism is cultured in a selective medium, the culture is irradiated with excitation light, and the intensity of fluorescence from the culture is measured. And a method for measuring the number of viable bacteria and identifying the bacteria by applying the measured value to a calibration curve of the number of viable bacteria prepared in advance for a known microorganism species and the fluorescence intensity corresponding to the number of viable bacteria. The microorganisms that are the subject of the present invention are limited to Escherichia coli, Salmonella, and Staphylococcus aureus. Further, a device for diluting a specimen containing the microorganism to be measured into a plurality of stages and adding a fluorescent probe, and a fluorescent light for irradiating the specimen before and after culturing with excitation light and measuring the intensity of fluorescence emitted from the specimen. A measuring device, an external storage device in which a calibration curve of the number of viable bacteria prepared in advance for a known microorganism species and a fluorescence intensity corresponding to the number of bacteria is stored as a database, and an external storage device based on a detection value of the fluorescence measuring device. Search and refer to the database of the storage device for data processing,
A viable cell count measurement / species identification system comprising a probe addition device and a device for controlling a fluorescence measurement device is also provided. The fluorescence measuring device includes at least a light emitting means for generating excitation light; a means for irradiating the object with the excitation light; a means for receiving fluorescence generated from the object to be measured; and the excitation of stray light entering together with the fluorescence. A band-pass filter that cuts light; and a photomultiplier tube that captures fluorescence passing through the band-pass filter. The means for receiving the fluorescence is, for example, an optical fiber.

[0008]

As described above, in the method of the present invention, the number of viable cells and the type of bacteria are identified by specializing in Escherichia coli, Salmonella, and Staphylococcus aureus. When a specific selection medium described in detail below is used to culture a sample suspected of containing the fungus, some grow by each bacteria and others do not. Therefore, it is possible to identify the microorganism species that has grown according to the type of the medium, that is, the microorganism species present in the sample.

Since the combination of the microorganism species and the medium is known in advance, the microorganism is cultured in this medium by a standard method, and the number of viable cells at each culturing time is counted by a known and established method. The fluorescence intensity corresponding to the number of bacteria is measured, a calibration curve is created, and stored as a database. If an unknown sample is cultured in a selective medium and grows, the bacterial species can be easily identified from the type of the medium. At the same time, if the fluorescence intensity at the time of the predetermined culture time is compared with the database of the corresponding medium / microorganism, the viable cell count of the microorganism can be immediately obtained.

[0010] When an optical fiber is used as the fluorescence receiving means, weak fluorescence can be caught as compared with the conventional dichroic mirror, and the measurement sensitivity increases.

[0011]

The present invention will be described in more detail with reference to the following examples. A feature of the method of the present invention lies in the use of the selective medium. As described above, the method of the present invention is used specifically for Escherichia coli, Salmonella, and Staphylococcus aureus. A medium capable of selectively growing only Escherichia coli and Salmonella has, for example, the following composition. K ₂ HPO ₄ 7 g / l, KH ₂ PO ₄ 3 g / l, MgSO ₄ .7H ₂ O 0.1 g / l, (NH ₄ ) ₂ SO ₄ 0.1 g / l, triptycase 1 g / l, glucose 2 g / l, this medium is called A medium. In this medium, only E. coli and Salmonella grow selectively. Therefore, if a specimen is inoculated and cultured in this medium and a colony appears, it can be estimated that the colony is caused by Escherichia coli and Salmonella.

A medium capable of selectively growing only Salmonella has, for example, the following composition. K ₂ HPO ₄ 7 g / l, KH ₂ PO ₄ 3 g / l, MgSO ₄ .7H ₂ O 0.1 g / l, (NH ₄ ) ₂ SO ₄ 0.1 g / l, triptycase 1 g / l, glucose 2 g / l, novobiocin qs This medium is called B medium. This medium is obtained by adding and mixing novobiocin to the above-mentioned medium A. Novobiocin is a macrolide antibiotic and is commercially available, for example, under the registered trademark "Albamycin" from Upjohn, USA. The A medium itself is a selective medium for Escherichia coli and Salmonella, but when novobiocin is added thereto, Escherichia coli does not grow, and only Salmonella grows selectively. Therefore, this medium is inoculated with the specimen, cultured,
If a colony appears, it can be presumed that it is due to Salmonella. The amount of novobiocin added is generally 5μ.
g / ml to 30 μg / ml, preferably 10 μg / m
1-20 μg / ml, most preferably 15 μg / ml
It is.

A medium capable of selectively growing only Staphylococcus aureus has, for example, the following composition. Trypticase 2.5 g / l, Phytone 0.5 g / l, NaCl 5 g / l, Tween 80 1.0 g / l, Azuleonam 0.25 μg / ml This medium is called C medium. Azuleonam is a "monobactam" antibiotic developed by Squibb, USA, which has specific activity against Escherichia coli and Salmonella, but has little activity against Staphylococcus aureus. Therefore, if a specimen is inoculated and cultured in this medium and a colony appears, it can be estimated that the colony is caused by Staphylococcus aureus.

Since the fluorescence intensity of a commercially available selection medium is extremely high, it is extremely difficult or impossible to detect the change in the fluorescence intensity of a culture by the method of the present invention. Among the medium components, meat extract, peptone, yeast extract and the like have extremely high fluorescence intensity. However, these components are generally nutrient components essential for the growth of the microorganism. According to the study of the present inventors, since the nutritional requirements of Escherichia coli and Salmonella are not high, it has been confirmed that they can grow even if these are removed. Therefore, it can be said that the medium A for Escherichia coli and Salmonella is a substantially non-fluorescent medium.

[0015] On the other hand, in the case of Staphylococcus aureus, the fluorescence from the culture alone cannot provide a sufficient measurement.
A fluorescent label called 0-10 dodecyl bromide is added and the fluorescence intensity is measured. The added A0-10 dodecyl bromide specifically binds to Staphylococcus aureus and shows a fluorescence intensity according to the number of bacteria. That is, A0-10 dodecyl bromide itself does not emit fluorescence, but emits fluorescence only when bound to S. aureus, so that it can be used as a fluorescent label in the method of the present invention.

Next, the present invention will be described in more detail with reference to the drawings. FIG. 1 is a schematic plan view of an example of a microplate 1 used to carry out the method of the present invention. The material of the plate itself is not particularly limited, but any material can be used as long as it scatters excitation light as little as possible and does not hinder the generation and reception of fluorescence. For example, a transparent polycarbonate can be used. However, it is a matter of course that the present invention is not limited to this. The plate shown is 9
It has six holes. The mode of use of the holes is not particularly limited.
As an example, as shown, the first of the 12 rows
Bacterial solutions or specimens are placed in the columns and the seventh column, and the second to sixth columns and the eighth to twelfth columns are used for serial dilution. In this example, the first to sixth columns are used as controls (for example, for measuring the background value of the fluorescence before culture), and the seventh to sixth columns are used.
The column and subsequent columns are used for fluorescence measurement after culture. Lines A to H can contain the same bacteria or medium,
It is also possible to divide the medium into rows A to D and rows E to H and to add each of the above-mentioned media. The specific manner of using such holes is well known to those skilled in the art and need not be further described.

Next, a series of procedures for carrying out the method of the present invention will be described with reference to FIG. First, FIG.
A microplate 1 as shown in (1) is prepared, and a predetermined selective medium is dispensed into a predetermined hole of the plate in a medium dispensing zone 2. It is well known to those skilled in the art that dispensing can be performed either manually or by an automatic machine. Next, in the bacterial solution dispensing zone 3, the specimen (solid or liquid) or the bacterial solution is adjusted / emulsified by a commercially available blender or stomacher, and is divided into the first and seventh columns of the plate as described above. Note. This dispensing can also be performed manually or by an automatic machine. However, although not particularly limited, medium dispensing and bacterial solution dispensing are not incorporated in the automatic viable cell count / species identification system of the present invention. Therefore,
It should be performed by a person using the system of the present invention as preprocessing.

Thereafter, the plate 1 is loaded on the serial dilution / fluorescence probe addition device 4 of the system of the present invention. Here, the bacterial liquids in the first and seventh columns are, for example,
10 μl are automatically collected and injected into the holes of the second and eighth rows, respectively. Next, 10 μl volumes were automatically collected from the holes in the second and eighth rows, respectively, and
Inject into each row of holes. Hereafter, this is the sixth column and 1
Repeat in order up to the second row. Then, 10 ⁰ , 1
Seven dilution series of 0 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 and 10 ^-6 are obtained. According to the experiments performed by the present inventors, it was confirmed that a dilution of 10 ⁻⁶ or more did not produce colonies and was meaningless.

When the serial dilution is completed, the AO-10 dodecyl bromide probe solution is dispensed only to all the wells on the control side of the plate. As mentioned above, the AO-10 dodecyl bromide probe is only specific for Staphylococcus aureus, but in order to assure background values,
It is preferably used also in the case of Escherichia coli and Salmonella.

After dispensing the probe solution into the control side hole of the plate, the plate is transferred to the fluorescence measuring device 5. Here, the fluorescence intensity before culturing is measured and stored in the external storage device of the data processing / control device 6 as a background value. Thereafter, the plate is transferred to the incubator 7 and cultured under predetermined conditions for a predetermined time. After the culturing, the plate is returned to the fluorescent probe adding device again, and the AO-10 dodecyl bromide probe solution is dispensed into all the holes on the culture side of the plate. To ensure the binding reaction between the bacteria and the probe, the plate is left for several minutes to 10 minutes after dispensing the probe. Thereafter, the fluorescence intensity on the culture side of the plate is measured by a fluorescence measurement device, and the measurement result is transmitted to an external storage device of the data processing / control device 6. The true fluorescence intensity is obtained by subtracting the background value before the culture from the fluorescence intensity after the culture. By comparing the result of the subtraction with a database in the external storage device, the bacterial species and the viable cell count are determined.

FIG. 3 is a flowchart showing the above processing procedure. After the pretreatment 10 of the medium dispensing and the bacterial solution dispensing, it enters the serial dilution / probe dispensing 11. Next, an initial fluorescence intensity measurement 12 is performed. The measured values are processed in line 13
It is transmitted to the control device 14. After the initial fluorescence intensity measurement, culture 14 is performed. After the culture, the process returns to step 11, and probe dispensing is performed again. After that, fluorescence intensity measurement 15 is performed after the culture. The measured values are transmitted on line 16 to the data processing and control unit 14. The data processing / control device 14 has a database 17 as an external storage device. The transmitted measured fluorescence value is collated with the database, the number of bacteria and the type of bacteria are determined, and output at 18.

FIG. 4 shows a schematic configuration of a viable cell count measurement / species identification system 20 of the present invention. As described above, the system of the present invention basically includes the serial dilution / fluorescence probe addition device 4, the fluorescence measurement device 5, and the data processing / control device 6. Although not specifically excluded, incubator 7 is generally not included in system 20 of the present invention. That is, the person who uses the system 20 of the present invention should prepare himself. In the illustrated embodiment, the serial dilution / fluorescence probe addition device 4 and the fluorescence measurement device 5 are as if they were separate devices.
It is obvious to those skilled in the art that both can be combined to form a single system. In the figure, the outline arrows I to IV indicate the transport direction of the plate, and I and IV can be performed by an automatic handling mechanism (not shown) in the system. II and III are manual.

The specific configuration of the serial dilution / fluorescence probe addition device 4 is not particularly limited. Any device configuration that can achieve the intended purpose of serial dilution and addition of a fluorescent probe may be used. As an example, as shown, X-
The YZ-Z 'axis moving mechanism 22 is used, the quad tip dispensing mechanism 24 is arranged on the Z' axis, and the probe liquid tank 26 can be provided. Although not shown, since the chips are disposable, a chip supply / discard mechanism can be provided. It is preferable that the serial dilution / fluorescence probe addition device is housed in a closed box, and the inside of the box is maintained in a sterile state by a UV (ultraviolet) sterilizing lamp 28.

The specific configuration of the fluorescence measuring device 5 is not particularly limited. However, in the system 20 of the present invention,
It is preferable to use a measuring device having an optical system as shown in FIG. The illustrated fluorescence measuring device 5 has, for example, an optical system unit 30 and an XY table 32 for moving a plate. The feature of the illustrated optical system unit 30 is that fluorescence is received by the optical fiber 36 held in the holder 34, which will be described in detail below. The fluorescence measurement device is also housed in a closed box,
It is preferable to maintain the inside of the box aseptically by a V (ultraviolet) sterilizing lamp 28. Further, as described above, the serial dilution / fluorescence probe addition device 4 and the fluorescence measurement device 5 can be accommodated in one closed box.

In the system 20 of the present invention, the serial dilution / fluorescence probe addition device 4 and the fluorescence measurement device 5 perform data processing and
It is connected to the control device 6. If necessary, they can be connected via the expansion slot box 40. The data processing and control device 6 also functions as a controller for the devices 4 and 5 so that the device 4 and the device 5 operate according to a procedure preprogrammed in the data processing and control device 6. The data processing / control device 6 may include an external storage device 42 such as an HDD for storing a database, in addition to the CPU in the main body. Further, a CRT 44 and a printer 46 for displaying control contents and outputting measurement results can be provided.

FIG. 5 shows a conceptual configuration diagram of an optical system in the fluorescence measuring device of the viable cell count measuring / species identification system of the present invention. As a light source, for example, a xenon lamp 50 is used, an auxiliary mirror 52 is arranged behind, a condenser lens 54 is arranged in front, and a heat ray absorbing filter 56 is arranged in front of the lens to constitute a light source system. doing. The light beam collimated by the condenser lens 54 is passed through an excitation light bandpass filter 58 in order to extract only excitation light having a specific wavelength. This filter is rotated by a motor so that excitation light of four wavelengths 0 to 3 can be obtained as shown in the figure. However, the number of filters is not limited. The wavelength of the excitation light itself is not an essential requirement of the present invention, but as a general index,
365 or 470 for E. coli and Salmonella
It is preferred to use excitation light with a wavelength of nm. On the other hand, in the case of Staphylococcus aureus, it is preferable to use excitation light having a wavelength of 490 nm. The excitation light that has passed through the bandpass filter 58 passes through the relay lens 62 and the variable stop 64. The collimated excitation light is stopped by the variable stop 64,
6 is refracted by 90 °. The refraction excitation light is applied to the imaging lens 68.
Focus on the wells of the microplate 7
The sample within 0 is irradiated. Alternatively, the excitation light exiting the excitation light bandpass filter 58 can be guided by an optical fiber (not shown) to irradiate the specimen.
If optical fiber is used, relay lens, variable aperture,
The need for a reflecting mirror and an imaging lens is eliminated, and the energy attenuation of the excitation light can be suppressed.

The fluorescence generated from the specimen is received by an optical fiber. The number of optical fibers for fluorescence reception may be one or more (for example, six). The optical fiber is preferably arranged so as to surround the specimen in a circular shape.
For this reason, it is preferable to hold by the holder 34 as shown simply in FIG. The holding angle of the optical fiber with respect to the vertically incident excitation light is not particularly limited. Any angle that can achieve the maximum efficiency for receiving the fluorescent light may be used. Such an angle can be easily determined by those skilled in the art.
Generally, 45 ° is preferred.

The fluorescence received by the optical fiber 72 is further guided to a fluorescence-side bandpass filter 74 and an excitation light-side bandpass filter 76. Filters 74 and 76
Must use the same numbered filter as filter 58. Although not shown, the filters 74 and 76 are simultaneously driven and controlled by the motor 60 at the same time as the filter 58. The fluorescence-side band-pass filter 74 should use a filter that passes fluorescence of 520 and 530 nm in the case of Escherichia coli and Salmonella, and a filter that can pass fluorescence of 535 and 540 nm in the case of Staphylococcus aureus. Is preferred. The filter 76 can be used to remove stray light excitation data from the data so as not to be a factor in disturbing the fluorescence intensity. But,
This filter 76 does not necessarily need to be used. Immediately after each filter, photomultipliers 78 and 80 are arranged. The electric signal from the photomultiplier is amplified by an amplifying circuit 82.
And 84 are transmitted to a data processing and control device as shown in FIGS.

Next, a method of creating a database will be described. Data on the viable cell count is obtained by the conventional agar plate method. For example, from a sample with a dilution ratio of 10 ^-6 times, 0.1
An agar plate is inoculated on the agar plate, and cultured at 37 ° C. in an air atmosphere for a predetermined time. Further, 0.1 ml is collected from a sample having a dilution factor of 10 ^-7 times, inoculated on an agar plate, and similarly cultured at 37 ° C. in an atmosphere in the atmosphere for a predetermined time. After culture
The number of colonies appearing on the agar plate is counted. For example,
In the case of 10 ^-6 samples, 100 colonies appeared and 10
^-7 sample, 10 colonies appeared, 10
^The number of bacteria in the sample of ^-6 was 100 × 10 ⁶ × 10 = 10
It becomes ⁹ / ml, in 10 ^-7 specimens, 10 × 10 ⁷ ×
10 = 10 ⁹ cells / ml. From this result, the number of viable bacteria in the sample is estimated to be 10 ⁹ . If this measurement is performed, for example, at 3, 4.5, 6, and 7.5 hours after the start of the culture, a characteristic curve showing the change in the number of bacteria over time can be obtained.

At the same time, the fluorescence measurement system of the present invention was used for 3 hours, 4.5 hours,
The fluorescence intensity at time 7.5 hours is measured. When a characteristic curve is created from this data, it can be confirmed that the fluorescence intensity increases over time. Therefore, by combining the data of the number of viable bacteria at each culture time with the data of the fluorescence intensity at each culture time, a calibration curve of the fluorescence intensity with respect to the number of viable bacteria can be created. This is stored and stored as a database. Therefore, if the species of the unknown sample is identified,
The number of viable cells can be obtained by applying the measured fluorescence intensity to the viable cell count / fluorescence intensity calibration curve database of the species.

The basic concept for creating a database for use in the method and system of the present invention is as described above. Therefore, the reliability of the method and system of the present invention is not remedied, but depends on the reliability of the database. For this reason, it is preferable to repeat as many culture experiments as possible, statistically process the obtained results, minimize variations, and construct a universal database as much as possible. For example, since Escherichia coli has many types of bacteria and mutants, even if the culture conditions and the fluorescence measurement conditions are the same, each of the bacteria or strains causes a slight change in the fluorescence intensity even with the same number of viable bacteria. . According to the method of the present invention, a specific strain in the coliform group cannot be identified. Therefore, even if various Escherichia coli strains are mixed in the sample, it is estimated as a general generic number of living coliform bacteria. The same applies to Salmonella and Staphylococcus aureus.

Since noise may greatly affect the predicted value of the initial bacterial concentration due to factors such as the above-mentioned differences in strains, the establishment of a firm database and the obtained fluorescence intensity Can be processed by fuzzy logic. A function that associates a specific variable value (such as a measured fluorescence intensity) with a fuzzy variable expressed by a language (such as a large fluorescence intensity) is called a membership function. In addition, a relation between events, such as "If the amount of change in fluorescence intensity is large, the initial bacterial concentration is extremely large", is called a rule base. According to the fuzzy theory, to estimate the initial bacterial concentration from the change in fluorescence intensity, a database of rules (that is, a rule base) for estimating the initial bacterial concentration from the change in fluorescence intensity for each identified bacterial species and dilution concentration is used. A membership function for each fuzzy variable is created and stored in the data processing device. According to this method, even if there is an error in the measured value of the fluorescence intensity, the deviation from the expected value is smoothed / corrected to some extent by the processing of “ambiguity” which is a characteristic of fuzzy, and the noise is canceled. Thus, the initial bacterial concentration can be measured with high reliability. With the accumulation of the rule base, the membership function, and the experimental data, it is possible to further improve the measurement accuracy by sequentially performing correction and tuning. In addition, by adding a learning function, these tunings can be automatically performed, and the function can be improved.

FIGS. 6 to 8 show an example of a calibration curve created based on the basic concept as described above. FIG. 6 is a graph showing the relationship between the fluorescence intensity and the number of viable cells in the culture of Escherichia coli (JCM1646 strain). The white hexagonal spot is a characteristic curve showing the time-dependent change in the number of viable bacteria with respect to the culture time. On the other hand, ● is a characteristic curve showing the change over time of the fluorescence intensity with respect to the culture time. Figure 7 shows Salmonella (S206 strain)
5 is a graph showing the relationship between the fluorescence intensity and the number of viable bacteria in the culture of Example 1. The white hexagonal spot is a characteristic curve showing the time-dependent change in the number of viable bacteria with respect to the culture time. On the other hand, ● is a characteristic curve showing the change over time of the fluorescence intensity with respect to the culture time. FIG. 8 is a graph showing the relationship between the fluorescence intensity and the viable cell count in the culture of Staphylococcus aureus (JCM2413 strain).
The white hexagonal spot is a characteristic curve showing the time-dependent change in the number of viable bacteria with respect to the culture time. On the other hand, ● is a characteristic curve showing the change over time of the fluorescence intensity with respect to the culture time.

[0034]

As described above, according to the method and system of the present invention, species of Escherichia coli, Salmonella or Staphylococcus aureus are identified within a short time of about 8 hours, and these bacteria are also identified. The number can be measured.

[Brief description of the drawings]

FIG. 1 is a schematic plan view of an example of a microplate used for implementing the method and system of the present invention.

FIG. 2 is a schematic conceptual diagram showing the progress of the process of the method of the present invention.

FIG. 3 is a flowchart showing a flow of a processing procedure of the method of the present invention.

FIG. 4 is a schematic configuration diagram of a system of the present invention.

FIG. 5 is a schematic configuration diagram of an optical system for fluorescence measurement used in the system of the present invention.

FIG. 6 is a characteristic curve showing the relationship between the fluorescence intensity and the number of viable cells in the culture of Escherichia coli.

FIG. 7 is a characteristic curve showing the relationship between the fluorescence intensity and the number of viable cells in the culture of Salmonella.

FIG. 8 is a characteristic curve showing the relationship between the fluorescence intensity and the number of viable cells in the culture of Staphylococcus aureus.

[Explanation of symbols]

 Reference Signs List 1 Microplate 2 Medium dispensing zone 3 Bacterial solution dispensing zone 6 Data processing / control device 7 Incubator

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takao Kutsuno 2-6-1 Otemachi, Chiyoda-ku, Tokyo Within Hitachi Electronics Engineering Co., Ltd. (72) Rika Kasuga 2-6-Otemachi, Chiyoda-ku, Tokyo 2 Hitachi Electronics Engineering Co., Ltd. (72) Inventor Shinpei Suzuki 2-6-2 Otemachi, Chiyoda-ku, Tokyo Hitachi Electronics Engineering Co., Ltd. (72) Fukuo Iwatani 2-6-Otemachi, Chiyoda-ku, Tokyo No. 2 Inside Hitachi Electronics Engineering Co., Ltd. (72) Inventor Isao Endo 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Teruyuki Nagato 2-1 Hirosawa, Wako-shi, Saitama Pref. Inventor Kazusa Asama 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN (72) Inventor Benno Yoshimi 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN (58) Field surveyed (Int. Cl. ⁶ , DB name) C12Q 1/04 C12M 1/34 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] An apparatus for diluting a specimen containing a microorganism to be measured into a plurality of stages and adding a fluorescent probe, irradiating the specimen before and after culturing with excitation light to reduce the intensity of the fluorescence emitted from the specimen. A fluorescence measurement device to be measured, an external storage device in which a calibration curve of the number of viable bacteria prepared in advance for a known microorganism species and the fluorescence intensity corresponding to the number of bacteria is stored as a database, and a detection value by the fluorescence measurement device. A system for measuring the number of viable bacteria and identifying bacteria species, comprising a device for searching and referencing a database in an external storage device based on the data processing, performing data processing, and controlling a dilution / probe adding device and a fluorescence measuring device. 2. A fluorescence measuring device comprising: at least a light emitting means for generating excitation light; a means for irradiating the object with the excitation light; a means for receiving fluorescence generated from the object to be measured; 2. The system according to claim 1, further comprising: a band-pass filter that cuts off the stray light excitation light coming in; and a photomultiplier tube that captures the fluorescence that has passed through the band-pass filter. 3. A fluorescence receiving means according an optical fiber
Item 2. The system of item 2 .